Hydroxyproline, an amino acid found almost exclusively in collagen, results from the enzymatic conversion of proline in peptide linkage rather than incorporation of this amino acid directly in growing nascent collagen peptides. Although prolyl hydroxylase the enzyme catalyzing this reaction had been thought to be a soluble cytoplasmic enzyme for over a decade recent evidence has indicated the particulate nature of this enzyme in a variety of tissues. We propose to localize the site of the hydroxylation of peptide bound proline during collagen biosynthesis in lung tissue by characterizing the particle containing the enzyme involved in prolyl hydroxylation. Furthermore we will determine if the substrate for prolyl hydroxylase is located in this subcellular fraction. We will also determine the relationship between particulate enzyme activity and the rate of peptidyl proline hydroxylation during collagen synthesis. The specific aims of these studies are: 1) To determine if protocollagen is hydroxylated at a site in the cell where particulate enzyme is found. 2) To identify the subcellular fraction containing enzyme activity. 3) To determine if the rate of prolyl hydroxylation of tissues is proportional to the activity of particulate enzyme. 4) To determine if this subcellular fraction is also the site of lysyl hydroxylation. 5) To add these subcellular fractions containing prolyl hydroxylase to an in vitro protein synthesizing system and synthesize collagen. 6) To determine of polysomes for collagen synthesis can be isolated from this subcellular fraction. 7) To isolate collagen mRNA and possible specific factors for the translation of collagen mRNA from this subcellular fraction.